Maize transformation: history, progress, and perspectives.

Maize functional genomics research and genetic improvement strategies have been greatly accelerated and refined through the development and utilization of genetic transformation systems. Maize transformation is a composite technology based on decades' efforts in optimizing multiple factors involving microbiology and physical/biochemical DNA delivery, as well as cellular and molecular biology. This review provides a historical reflection on the development of maize transformation technology including the early failures and successful milestones. It also provides a current perspective on the understanding of tissue culture responses and their impact on plant regeneration, the pros and cons of different DNA delivery methods, the identification of a palette of selectable/screenable markers, and most recently the development of growth-stimulating or morphogenic genes to improve efficiencies and extend the range of transformable genotypes. Steady research progress in these interdependent components has been punctuated by benchmark reports celebrating the progress in maize transformation, which invariably relied on a large volume of supporting research that contributed to each step and to the current state of the art. The recent explosive use of CRISPR/Cas9-mediated genome editing has heightened the demand for higher transformation efficiencies, especially for important inbreds, to support increasingly sophisticated and complicated genomic modifications, in a manner that is widely accessible. These trends place an urgent demand on taking maize transformation to the next level, presaging a new generation of improvements on the horizon. Once realized, we anticipate a near-future where readily accessible, genotype-independent maize transformation, together with advanced genomics, genome editing, and accelerated breeding, will contribute to world agriculture and global food security.

Plastid Transformation: How Does it Work? Can it Be Applied to Crops? What Can it Offer?

In recent years, plant genetic engineering has advanced agriculture in terms of crop improvement, stress and disease resistance, and pharmaceutical biosynthesis. Cells from land plants and algae contain three organelles that harbor DNA: the nucleus, plastid, and mitochondria. Although the most common approach for many plant species is the introduction of foreign DNA into the nucleus (nuclear transformation) via Agrobacterium- or biolistics-mediated delivery of transgenes, plastid transformation offers an alternative means for plant transformation. Since there are many copies of the chloroplast genome in each cell, higher levels of protein accumulation can often be achieved from transgenes inserted in the chloroplast genome compared to the nuclear genome. Chloroplasts are therefore becoming attractive hosts for the introduction of new agronomic traits, as well as for the biosynthesis of high-value pharmaceuticals, biomaterials and industrial enzymes. This review provides a comprehensive historical and biological perspective on plastid transformation, with a focus on current and emerging approaches such as the use of single-walled carbon nanotubes (SWNTs) as DNA delivery vehicles, overexpressing morphogenic regulators to enhance regeneration ability, applying genome editing techniques to accelerate double-stranded break formation, and reconsidering protoplasts as a viable material for plastid genome engineering, even in transformation-recalcitrant species.

Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2.

KEY MESSAGE: Discriminatory co-expression of maize BBM and WUS transcriptional factor genes promoted somatic embryogenesis and efficient Agrobacterium -mediated transformation of recalcitrant maize inbred B73 and sorghum P898012 genotypes without use of a selectable marker gene. The use of morphogenic regulators to overcome barriers in plant transformation is a revolutionary breakthrough for basic plant science and crop applications. Current standard plant transformation systems are bottlenecks for genetic, genomic, and crop improvement studies. We investigated the differential use of co-expression of maize transcription factors BABY BOOM and WUSCHEL2 coupled with a desiccation inducible CRE/lox excision system to enable regeneration of stable transgenic recalcitrant maize inbred B73 and sorghum P898012 without a chemical selectable marker. The PHP78891 expression cassette contains CRE driven by the drought inducible maize RAB17M promoter with lox P sites which bracket the CRE, WUS, and BBM genes. A constitutive maize UBI M promoter directs a ZsGreen GFP expression cassette as a reporter outside of the excision sites and provides transient, transgenic, and developmental analysis. This was coupled with evidence for molecular integration and analysis of stable integration and desiccation inducible CRE-mediated excision. Agrobacterium-mediated transgenic introduction of this vector showed transient expression of GFP and induced somatic embryogenesis in maize B73 and sorghum P898012 explants. Subjection to desiccation stress in tissue culture enabled the excision of CRE, WUS, and BBM, leaving the UBI M::GFP cassette and allowing subsequent plant regeneration and GFP expression analysis. Stable GFP expression was observed in the early and late somatic embryos, young shoots, vegetative plant organs, and pollen. Transgene integration and expression of GFP positive T0 plants were also analyzed using PCR and Southern blots. Progeny segregation analysis of primary events confirmed correlation between functional GFP expression and presence of the GFP transgene in T1 plants generated from self pollinations, indicating good transgene inheritance. This study confirms and extends the use of morphogenic regulators to overcome transformation barriers.

The WOX Genes from the Intermediate Clade: Influence on the Somatic Embryogenesis in Medicago truncatula.

Transcription factors from the WOX family are well-known regulators of cell proliferation and differentiation in plants. Herein, we focused on several WOX genes from the intermediate clade and checked their impact on somatic embryogenesis using the model legume object Medicago truncatula. As a result, we show that MtWOX9-1 overexpression not only stimulates somatic embryogenesis in the embryogenic M. truncatula line, as it was shown previously, but can also induce somatic embryogenesis in the non-embryogenic line. Other intermediate clade WOX, including the close paralog of MtWOX9-1, as well as WOX11 homologs, did not have any significant impact on somatic embryogenesis in our in vitro cultivation system. Together, our results give new information about the diversity of the WOX family proteins and their specific functions. These data can be used for the search of new regeneration stimulators.

Genetic Transformation of Apomictic Grasses: Progress and Constraints.

The available methods for plant transformation and expansion beyond its limits remain especially critical for crop improvement. For grass species, this is even more critical, mainly due to drawbacks in in vitro regeneration. Despite the existence of many protocols in grasses to achieve genetic transformation through Agrobacterium or biolistic gene delivery, their efficiencies are genotype-dependent and still very low due to the recalcitrance of these species to in vitro regeneration. Many plant transformation facilities for cereals and other important crops may be found around the world in universities and enterprises, but this is not the case for apomictic species, many of which are C4 grasses. Moreover, apomixis (asexual reproduction by seeds) represents an additional constraint for breeding. However, the transformation of an apomictic clone is an attractive strategy, as the transgene is immediately fixed in a highly adapted genetic background, capable of large-scale clonal propagation. With the exception of some species like Brachiaria brizantha which is planted in approximately 100 M ha in Brazil, apomixis is almost non-present in economically important crops. However, as it is sometimes present in their wild relatives, the main goal is to transfer this trait to crops to fix heterosis. Until now this has been a difficult task, mainly because many aspects of apomixis are unknown. Over the last few years, many candidate genes have been identified and attempts have been made to characterize them functionally in Arabidopsis and rice. However, functional analysis in true apomictic species lags far behind, mainly due to the complexity of its genomes, of the trait itself, and the lack of efficient genetic transformation protocols. In this study, we review the current status of the in vitro culture and genetic transformation methods focusing on apomictic grasses, and the prospects for the application of new tools assayed in other related species, with two aims: to pave the way for discovering the molecular pathways involved in apomixis and to develop new capacities for breeding purposes because many of these grasses are important forage or biofuel resources.

Morphogenic Regulators and Their Application in Improving Plant Transformation.

Generation of plant lines with transgene or edited gene variants is the desired outcome of transformation technology. Conventional DNA-based plant transformation methods are the most commonly used technology but these approaches are limited to a small number of plant species with efficient transformation systems. The ideal transformation technologies are those that allow biotechnology applications across wide genetic background, especially within elite germplasm of major crop species. This chapter will briefly review key regulatory genes involved in plant morphogenesis with a focus on in vitro somatic embryogenesis and their application in improving plant transformation.

Edit at will: Genotype independent plant transformation in the era of advanced genomics and genome editing.

The combination of advanced genomics, genome editing and plant transformation biology presents a powerful platform for basic plant research and crop improvement. Together these advances provide the tools to identify genes as targets for direct editing as single base pair changes, deletions, insertions and site specific homologous recombination. Recent breakthrough technologies using morphogenic regulators in plant transformation creates the ability to introduce reagents specific toward their identified targets and recover stably transformed and/or edited plants which are genotype independent. These technologies enable the possibility to alter a trait in any variety, without genetic disruption which would require subsequent extensive breeding, but rather to deliver the same variety with one trait changed. Regulatory issues regarding this technology will predicate how broadly these technologies will be implemented. In addition, education will play a crucial role for positive public acceptance. Taken together these technologies comprise a platform for advanced breeding which is an imperative for future world food security.

Transformation of Recalcitrant Sorghum Varieties Facilitated by Baby Boom and Wuschel2.

Most reliable transformation protocols for cereal crops, including sorghum (Sorghum bicolor L. Moench), rely on the use of immature embryo explants to generate embryogenic callus cells that are then transformed using Agrobacterium- or particle-bombardment-mediated DNA delivery. Subsequent to DNA transfer, most protocols rely on selectable markers for the recovery of stably transformed callus that is then regenerated to produce T0 plants. However, these protocols require specific genotypes that are innately capable of efficient embryogenic callus initiation. Here, we describe a system that makes use of the differential expression of the morphogenic regulators Baby Boom (Bbm) and Wuschel2 (Wus2) to achieve transformation in varieties of sorghum typically recalcitrant to standard transformation methods. © 2018 by John Wiley & Sons, Inc.